CN105198990A - Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses an antibody for broad-spectrum detection of Bt Cry1 toxoids as well as a preparation method and application thereof. The antibody is generated by secretion of hybridoma cells with the collection number of CCTCC NO:C2015145, obtained after coupling antigen polypeptide SEQ ID NO.2 and carrier protein KLH and then immunizing Balb/c mice. The ratio of the antibody to OD450 of six kinds of Bt Cry1 toxoids to negative holes (PBS solution) is far larger than 2.1, and the antibody has relatively high titer, has a broad-spectrum detection effect on the Bt Cry1 toxoids and makes up the blank of the broad-spectrum detection field of the Bt Cry1 toxoids.

Description

For antibody that Bt Cry1 toxoid wide spectrum detects and preparation method thereof and application

Technical field

The present invention relates to biochemistry, particularly a kind of antibody for the detection of BtCry1 toxoid wide spectrum and preparation method thereof and application.

Background technology

From the reported first such as Hilder in 1987 succeed in developing turn bacillus thuringiensis ( bacillusthuringiensis, Bt) since toxin gene zoophobous, have at present and be cloned more than 400 Bt toxin genes and check order, commercial formulation can be used as or turn existing tens classes of BtCry family gene crop (to comprise Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry2Aa, Cry2Ab, Cry3A, Cry3B, Cry34, Cry35 etc.), turn Bt toxin gene insect-resistance crop constantly occur and obtain large-area popularization, create extremely significant economical, society and ecological benefits, but along with the large-scale promotion of transgenic Bt plants, it receives publicity gradually to ecological safety risk and to the mankind and other mammiferous potential safety hazard.At present, repeatedly be in the news in China's rice and found BtCry toxin gene and toxin production, and serious detrimentally affect is created to China's rice outlet, along with greatly developing of transgenic technology, the BtCry toxin gene kind be employed is more and more many, the government safety supervision brought and the limitation of the technology of food processing enterprises raw material management and control more and more obvious, especially time gene test can not obtain promising result in for some netically modified foods, contratoxin expression product safe screen check survey particularly urgent.

The current detection method for toxin protein in transgenic product mainly adopts the antibody test technology for single toxin, owing to needing to prepare corresponding antibody for different toxin, and antibody preparation process is tediously long, cost is high, the requirement that wide spectrum examination detects can not be met.Therefore, the similar three-dimensional conformation feature how utilizing BtCry toxin to have, the BtCry toxin wide spectrum screening immunoassay technology developed based on general character structure is the work having very much using value.

Certain cross reaction is all there is in the monoclonal antibody according to bibliographical information about 90% for the antigen with general character structure, and for the toxin that other homology is higher, also there is certain cross reaction for single, the polyclonal antibody of a certain Bt Mycotoxin identification or single-chain antibody, although this has larger difference based on different Cry toxin aminoacid sequences, but the principle that its three-dimensional structure is closely similar, at present, there is not yet relevant report for BtCry1 toxoid detection wide spectrum antibody.

Summary of the invention

For the problems referred to above, the present invention is by mouse immune, and obtain for BtCry1 toxoid detection wide spectrum antibody, the present invention is achieved in that

For the antibody that BtCry1 toxoid wide spectrum detects, it is characterized in that, described antibody is that the hybridoma being CCTCCNO:C2015145 by preserving number secretes generation.

One strain preserving number is the hybridoma of CCTCCNO:C2015145, and the antibody of its secretion is used for BtCry1 toxoid wide spectrum and detects.

Further, hybridoma of the present invention obtains like this:

1) utilize MBS method synthetic immunogen, what be about to wait mole number is SEQIDNO.2 through KLH-NH2 and the aminoacid sequence of desalting treatment polypeptide mixes, and 4 DEG C are reacted 2h, and be then placed in PBS solution and dialyse 72h, adaptive immune is former;

2) be the immunogen of 1mg/ml and the 1:1 ratio mixing by volume of Fu Shi Freund's complete adjuvant by concentration, with 100ug/ dose immunization mouse only; Once every two weeks booster immunizations, replace Freund's complete adjuvant to mix with immunogen during booster immunization with Freund's incomplete adjuvant, after the 4th immunity terminates, blood sampling survey in a week is tired, and tires and reaches serum-dilution 10000 times, OD later ₄₅₀when value is greater than 1.0, carry out cytogamy with murine myeloma cell SP2/0; Then adopt indirect elisa method screening cells and supernatant, select positive colony hybridoma wells to carry out subclone, and with limiting dilution assay continuous cloning 2-3 time, namely obtain described hybridoma.

The application of antibody of the present invention in BtCry1 toxoid wide spectrum detects.

Further, application of the present invention, is specially:

A) be that the hybridoma of CCTCCNO:C2015145 is inoculated in Mice Body by preserving number, to induce legal system in body for ascites, then utilize caprylic acid-ammonium purifying ascites, obtaining concentration is the antibody of 25mg/ml, then with PBS solution by antibody dilution 1000-16000 doubly;

B) antibody is coated in solid phase carrier, 4 ° of C spend the night; Next day, PBST washed plate, added the MPBS of 3%, 200 μ L/ holes, and 37 ° of C hatch 2h; PBST washes plate, adds product to be detected, and 100 μ L/ holes, hatch 1h; Add enzyme labelled antibody and hatch 1h; PBST washes plate, adds substrate TMB-Urea Peroxide, 100 μ L/ holes, and after 37 ° of C hatch 15min, every hole adds 50 μ L concentration is 2mol/LH ₂sO ₄, terminate reaction; Simultaneously using PBS solution as negative control;

C) assaying reaction liquid OD ₄₅₀value, if the OD of product to be detected and negative control ₄₅₀ratio is greater than 2.1, then kind to be detected contains BtCry1 toxoid.

The present invention is according to Cry1Aa, Cry1Ab, the aminoacid sequence of Cry1Ac, Cry1B, Cry1C and Cry1F, from primary sequence similarity analysis, secondary antigen and hydrophilicity analysis and three-dimensional structural analysis, the antigenic peptide SEQIDNO.2 of design, after itself and carrier proteins KLH are carried out coupling, immune Balb/c mouse, have successfully been obtained the hybridoma cell strain can secreted for BtCry1 toxoid detection broad-spectrum monoclonal antibody, this cell strain preserving number is CCTCCNO:C2015145; Experiment shows, concentration is after this antibody dilution 16000 times of 25mg/ml, to six kinds of anatoxic OD of BtCry1 ₄₅₀the ratio of value and negative hole (PBS solution) is all much larger than 2.1, and it tires higher as seen, and possesses broad spectrum Detection results to BtCry1 toxoid, makes up the blank of BtCry1 toxoid wide spectrum detection field.

Accompanying drawing explanation

Fig. 1 is Amino acid sequences alignment's result schematic diagram of selected peptide section;

Fig. 2 is antigenicity and the hydrophilicity analysis result schematic diagram of selected peptide section;

Fig. 3 is that the 3 d structure model of six kinds of toxin carries out overlapping result schematic diagram;

Fig. 4 is the three-dimensional structure schematic diagram of selected peptide section;

Fig. 5 is monoclonal antibody ELISA detected result schematic diagram.

Embodiment

The preparation method of solution is related in embodiment:

(1) saturated ammonium sulphate solution:

In 500mL distilled water, add 500g ammonium sulfate, be heated to dissolve completely; Ambient temperature overnight, crystallization is let alone to stay in bottle, gets required amount before use, adjusts pH to 7.8 with NaOH;

(2) acetate buffer solution (0.06mol/LpH4.8):

NaAc0.29g, HAc0.141mL are dissolved in deionized water, are settled to 100mL, adjust pH to 4.8.

(3) phosphate buffered saline buffer (PBS, pH7.4):

NaCl8.0g, KCl0.2g, Na ₂hPO412H ₂o2.9g, KH ₂pO ₄0.2g, adding distil water is settled to 1L;

(4) washings (PBST): the PBS containing 0.05%Tween;

(5) confining liquid (MPBS): the PBS containing 3% skim-milk;

(6) citrate buffer (CPBS, substrate buffer solution, pH5.5):

C ₆h ₇o ₈(citric acid) 21g, Na ₂hPO ₄12H ₂o71.6g, adding distil water is settled to 1L;

(7) TMB and 25uL0.65%H of substrate: 10mg/mL ₂o ₂be dissolved in 9.875mLCPBS, now with the current;

(8) tetramethyl benzidine (TMB) mother liquor:

Take 10mg tetramethyl benzidine and be dissolved in 1mL dimethyl sulfoxide (DMSO), 4 DEG C of preservations;

(9) stop buffer (2MH ₂sO ₄):

The vitriol oil (18M) getting 11.8mL98% is dissolved in 80mL distilled water, and after cooling, constant volume is to 100mL;

(10) carbonate buffer solution (CBS):

Na ₂cO ₃1.59g; NaHCO ₃2.93g, constant volume is to 1L;

Involved reagent in embodiment:

Not formula Freund's complete adjuvant and Fu Shi Freund's incomplete adjuvant are all purchased from Sigma company;

Cry1Aa and Cry1B antibody is purchased from You Long bio tech ltd, Shanghai;

Aminoacid sequence involved in embodiment:

SEQIDNO.1:SFREWEADPTNPALREEMRI；

SEQIDNO.2：CSFREWEADPTNPALREEMRI。

Embodiment 1 prepares monoclonal antibody

(1) in GenBank database, Cry1Aa is retrieved, the aminoacid sequence of Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F;

(2) utilize DNAMAN software to be contrasted by the aminoacid sequence of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F, find out some peptide sequences that similarity is the highest, Amino acid sequences alignment's result of selected peptide section as shown in Figure 1;

(3) utilize DNAStar software to find out Cry1Aa, antigenicity and the stronger part of wetting ability in Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F, the antigenicity of peptide section and hydrophilicity analysis result are as shown in Figure 2;

(4) SWISS-MODEL Website construction Cry1Aa is finally utilized, Cry1Ab, Cry1Ac, Cry1B, the 3 d structure model of Cry1C and Cry1F six kinds of toxin, analyze 3 d structure model with Swiss-PdbViewer4.1.0 software, find out the region of six kinds of toxin three-dimensional structure overlaps, the 3 d structure model of six kinds of toxin carries out overlapping result as shown in Figure 3;

(5) primary sequence similarity analysis result, secondary antigen and hydrophilicity analysis result and three-dimensional structural analysis result are carried out Integrated comparative, Selective sequence similarity is higher, comparatively strong with wetting ability, that space conformation the is similar polypeptide fragment of antigenicity is the antigenic peptide most possibly producing broad spectrum immune effect, as immune haptens, this peptide section three-dimensional structure as shown in Figure 4, in Fig. 4, A is selected peptide section, and its aminoacid sequence is as shown in SEQIDNO.1; Transfer to Nanjing Genscript Biotechnology Co., Ltd. to synthesize this sequence, in order to carrier protein KLH, add Cys residue in one end of peptide chain, section of synthesized peptide sequence is as shown in SEQIDNO.2;

(6) immunogen preparation, with hemocyanin (KLH) for carrier proteins, adopt MBS method synthetic immunogen, concrete steps are as follows: take 5mgKLH-NH ₂be dissolved in the PBS of 1ml0.1mM, then add the Sulfo-MB that final concentration is 1mM, at 4 DEG C, react 2h; Carry out desalination after G25Sephadex gel permeation chromatography post and remove unnecessary linking agent;

Then by the KLH-NH after desalting treatment ₂with wait the polypeptide SEQIDNO.2 of mole number mix, after reacting 2h in 4 DEG C, then at 4 DEG C of temperature, be placed in PBS solution and dialyse 72h, change liquid once every 9-12h between dialysis period, namely adaptive immune is former, then packing, frozen for subsequent use in-20 DEG C;

(7) preparation of monoclonal antibody: Balb/c female mice 4 about the 8 week age of immunogen immune utilizing step (6) to obtain, during first immunisation, be after the immunogen of 1mg/ml and the 1:1 ratio mixing by volume of Fu Shi Freund's complete adjuvant by concentration, with the mode immune mouse of abdominal injection, immunizing dose is 100ug/;

Later every two weeks booster immunizations once, replace Freund's complete adjuvant mix with immunogen during booster immunization with Freund's incomplete adjuvant, the 4th the immunity survey that terminates to take a blood sample for a week is afterwards tired, and tires and reaches ideal value (that is: serum-dilution 10000 times and OD ₄₅₀value is greater than 1.0) time, again conveniently hybridoma fusion techniques (see Wu Jianxiang etc., " development of Pyricularia oryzae monoclonal antibody and the shadow noon to note fields thereof " microorganism journal, 2000,40 (6): 638-645) cytogamy is carried out with murine myeloma cell SP2/0, adopt indirect elisa method screening cells and supernatant, positive colony hybridoma is selected to carry out subclone, and clone 2-3 time continuously with limiting dilution assay, obtain the hybridoma cell strain of a strain stably excreting antibody.

Indirect elisa method step is as follows:

1. wrap quilt: with coating buffer CBS by coating antigen Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F are diluted to the concentration of 4 μ g/mL, join in 96 hole enzyme plates respectively, every hole 100 μ L, 4 ° of C bags are spent the night;

2. close: PBST washes plate three times, adds the MPBS of 3%, 200 μ L/ holes, put in 37 ° of C incubators and hatch 2h;

3. culture supernatant is added: PBST washes plate three times, draw the culture supernatant 100 μ L of cell hole to be detected, join in 96 hole enzyme plates, put in 37 ° of C incubators and hatch 1h, establish negative serum and positive serum (1/2000 application of sample) and acellular hole culture supernatant to contrast simultaneously;

4. add ELIAS secondary antibody: PBST washes plate three times, add the sheep anti-mouse igg (purchased from KPL company, working concentration 1/5000, diluent is MPBS) of HRP mark, every hole 100 μ L, puts in 37 ° of C incubators and hatches 1h;

5. develop the color: PBST washes plate four times, adds substrate (TMB-Urea Peroxide), and every hole 100 μ L, hatches 15min in 37 ° of C incubators;

6. stop: add 2mol/LH ₂sO ₄, every hole 50 μ L;

7. result judges: measure OD by microplate reader ₄₅₀value, the hole of P/N>=2.1 is judged to positive hole.

Applicant by obtain hybridoma cell strain from called after hybridoma cell strain DS2D3, and be preserved in China typical culture collection center (ChinaCenterforTypeCultureCollection on September 16th, 2015, CCTCC), address: Wuhan City, Hubei Province Wuhan University, postcode 430072, deposit number is CCTCCNO:C2015145.

Embodiment 2 antibody sensitivity technique

(1) to the hybridoma cell strain that mouse Inoculation embodiment 1 obtains, utilize in body and induce legal system for ascites, concrete steps are as follows:

By well-grown hybridoma gently purge get off, collected by centrifugation, with the resuspended counting of RPMI-1640 basic culture solution, cell quantity controls 1 ~ 2 × 10 ⁶within the scope of individual/mL, the mouse that abdominal injection uses paraffin oil pretreated in advance, every strain cell infusion 2 mouse, every mouse 0.5mL.The healthy state of close observation mouse and ascites sign, about about 10d, mouse web portion starts to expand, and when ascites is many as far as possible, draws neck to put to death mouse, absorption ascites of cutting open the belly.

By the ascites centrifugal 10min of 5000rpm under 4 ° of C collected, except degrease and hemocyte etc., in ascites after centrifugation, add equivalent glycerine, in-20 ° of C Refrigerator stores.

(2) ascites step (1) obtained adopts caprylic acid-ammonium to carry out purifying, and purification step is as follows:

Ascites 4 ° of centrifugal 15min of C12,000rpm, collect supernatant, abandon precipitation; 1 part of ascites fully mixes with 2 ~ 4 parts of acetate buffers, adjust pH to 4.5 ~ 4.8; Slowly add n-caprylic acid 33 μ L/mL ascites under stirring, under room temperature, mix 30min, rearmounted 4 ° of C leave standstill more than 2h; 4 ° of centrifugal 5min of C12,000rpm, collect supernatant, abandon precipitation; Supernatant adds the PBS damping fluid of the 0.1mol/LpH value 7.4 of 1/10 volume after filtering by double-layer filter paper, with 2mol/LNaOH adjust pH to 7.4, and 4 ° of C precoolings, in 30min, adding ammonium sulfate to final concentration under condition of ice bath is 0.277g/mL, and 4 ° of C leave standstill 2h; 4 ° of centrifugal 30min of C12,000rpm, abandon supernatant; With the 0.01mol/LpH value 7.4PBS dissolution precipitation of 1/10 former ascites volume; Dialyse one day to the PBS of 0.01mol/LpH value 7.4, centrifugally remove insoluble impurity, the settled solution obtained is antibody-solutions, and its concentration is 25mg/ml, is placed in-80 ° of C frozen;

(3) mensuration of antibody sensitivity: adopt indirect elisa method to measure.

Coating antigen adopts Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F six kinds of standard C ry1 toxoids, and concentration is 4ug/ml;

Antibody is the concentration that step (2) obtains is 25mg/ml antibody-solutions, and antibody is diluted 1000 times, 2000 times, 4000 times, 8000 times, 16000 times with PBS damping fluid successively;

Concrete steps are as follows:

1. wrap quilt: be coated in by antigens c ry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxin protein in 96 hole enzyme plates respectively, 100 μ L/ holes, 4 ° of C bags are spent the night;

2. close: PBST washes plate three times, adds the MPBS of 3%, 200 μ L/ holes, put in 37 ° of C incubators and hatch 2h;

3. add antibody: PBST washes plate three times, adds the monoclonal antibody after dilution respectively by 100uL/ hole, replace monoclonal antibody as negative control using PBS simultaneously;

4. add ELIAS secondary antibody: PBST washes plate three times, add the sheep anti-mouse igg (purchased from KPL company, by the dilution proportion of MPBS according to 1:5000) of HRP mark, every hole 100 μ L, puts in 37 ° of C incubators and hatches 1h;

5. develop the color: PBST washes plate four times, adds substrate (TMB-Urea Peroxide), and every hole 100 μ L, hatches 15min in 37 ° of C incubators;

6. stop: add 2mol/LH ₂sO ₄, every hole 50 μ L;

7. result judges: measure OD by microplate reader ₄₅₀value;

As shown in Figure 5, as seen from Figure 5, negative control value is all less than 0.1 to antibody titer measurement result, when antibody dilution 16000 times, to the OD of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F ₄₅₀value is respectively 0.78,0.535,0.688,0.764,0.744 and 0.71, with the ratio of negative hole all much larger than 2.1, shows that tiring of monoclonal antibody is higher, can be applied to the anatoxic broad spectrum of BtCry1 and detect.

Embodiment 3BtCry1 toxoid detection experiment

Test is divided into following four groups:

Experimental group: the antibody that embodiment 2 step (2) obtains, its concentration is 25mg/ml, with PBS solution 16000 times dilution;

Control group 1:Cry1Aa antibody, is diluted to and experimental group same concentrations with PBS;

Control group 2:Cry1B antibody, is diluted to and experimental group same concentrations with PBS;

Negative control: PBS solution;

Detecting step is as follows:

1. wrap quilt: each group of antibody is coated in solid phase carrier respectively, 100 μ L/ holes, 4 ° of C bags are spent the night, and make formation insolubilized antibody;

2. close: PBST washes plate three times, adds the MPBS of 3%, 200 μ L/ holes, put in 37 ° of C incubators and hatch 2h;

3. add antigen: PBST washes plate three times, adds the antigen of concentration 2ug/ml respectively, 100 μ L/ holes, hatch 1h, and the antibody on antigen and solid phase carrier is fully reacted, and form solid phase antigen antibody complex;

4. add enzyme labelled antibody: PBST washes plate three times, remove other non-binding substance, add enzyme labelled antibody, 100 μ L/ holes, hatch 1h, make formation insolubilized antibody determined antigen-enzyme labelled antibody sandwich complex, and washing removes non-desmoenzyme labeling antibody;

Enzyme labelled antibody is that (immunization method is identical with embodiment 1 step (7) monoclonal antibody for the immune rabbit polyclonal antibody prepared of mixed C ry1 toxoid, immunizing dose is that every rabbit often plants each 100ug of toxin, 600ug/ only altogether, and after three immunity, rabbit blood sampling detects, tire after reaching ideal value, heart adopts whole blood), after saturated ammonium sulphate purifying, mark with HRP, enzyme labelled antibody concentration is 20mg/ml, presses 1:4000 dilution during use with PBS;

5. develop the color: PBST washes plate four times, adds substrate (TMB-Urea Peroxide), and every hole 100 μ L, hatches 15min in 37 ° of C incubators;

6. stop: add 2mol/LH ₂sO ₄, every hole 50 μ L;

7. result judges: measure OD by microplate reader ₄₅₀value,

Respectively by OD that experimental group and control group 1,2 obtain ₄₅₀value and negative control compare, and ratio is greater than 2.1, and be designated as " √ ", otherwise be designated as "×", detected result is as shown in table 1:

Table 1:BtCry1 toxoid ELISA detected result

Group

Cry1Aa

Cry1Ab

Cry1Ac

Cry1B

Cry1C

Cry1F

Experimental group

√

√

√

√

√

√

Control group 1

√

×

×

×

×

×

Control group 2

×

×

×

√

×

×

From table 1, commercial antibody only can detect the BtCry1 toxoid of correspondence, and cannot detect other toxin; And the antibody that the present invention obtains, after reacting with six kinds of BtCry1 toxoid antigen, be all greater than 2.1 with the ratio of negative hole, can be used for BtCry1 toxoid wide spectrum and detect.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

SEQUENCELISTING

<110> Jiangsu Province farm institute

<120> is used for antibody of BtCry1 toxoid wide spectrum detection and preparation method thereof and application

<130>2

<160>2

<170>PatentInversion3.3

<210>1

<211>20

<212>PRT

<213> synthetic

<400>1

SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu

151015

GluMetArgIle

20

<210>2

<211>21

<212>PRT

<213> synthetic

<400>2

CysSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArg

151015

GluGluMetArgIle

20

Claims (5)

1. for the antibody that BtCry1 toxoid wide spectrum detects, it is characterized in that, described antibody is that the hybridoma being CCTCCNO:C2015145 by preserving number secretes generation.

2. a strain preserving number is the hybridoma of CCTCCNO:C2015145, and the antibody of its secretion is used for BtCry1 toxoid wide spectrum and detects.

3. according to claim 1 for the antibody that BtCry1 toxoid wide spectrum detects, it is characterized in that, described hybridoma obtains like this:

1) utilize MBS method synthetic immunogen, be about to the KLH-NH through desalting treatment waiting mole number ₂the polypeptide being SEQIDNO.2 with aminoacid sequence mixes, 4 DEG C of reaction 2h, and be then placed in PBS solution and dialyse 72h, adaptive immune is former;

2) be the immunogen of 1mg/ml and the 1:1 ratio mixing by volume of Fu Shi Freund's complete adjuvant by concentration, with 100ug/ dose immunization mouse only; Once every two weeks booster immunizations, replace Freund's complete adjuvant to mix with immunogen during booster immunization with Freund's incomplete adjuvant, after the 4th immunity terminates, blood sampling survey in a week is tired, and tires and reaches serum-dilution 10000 times, OD later ₄₅₀when value is greater than 1.0, carry out cytogamy with murine myeloma cell SP2/0; Then adopt indirect elisa method screening cells and supernatant, select positive colony hybridoma wells to carry out subclone, and with limiting dilution assay continuous cloning 2-3 time, namely obtain described hybridoma.

4. the application of antibody in BtCry1 toxoid wide spectrum detects as claimed in claim 1.

5. apply according to claim 4, it is characterized in that, concrete steps are:

A) be that the hybridoma of CCTCCNO:C2015145 is inoculated in Mice Body by preserving number, to induce legal system in body for ascites, then utilize caprylic acid-ammonium purifying ascites, obtaining concentration is the antibody of 25mg/ml, then with PBS solution by antibody dilution 1000-16000 doubly;

B) antibody after dilution is coated in solid phase carrier, 4 ° of C spend the night; Next day, PBST washed plate, added the MPBS of 3%, 200 μ L/ holes, and 37 ° of C hatch 2h; PBST washes plate, adds product to be detected, and 100 μ L/ holes, hatch 1h; Add enzyme labelled antibody and hatch 1h; PBST washes plate, adds substrate TMB-Urea Peroxide, 100 μ L/ holes, and after 37 ° of C hatch 15min, every hole adds 50 μ L concentration is 2mol/LH ₂sO ₄, terminate reaction; Simultaneously using PBS solution as negative control;

C) assaying reaction liquid OD ₄₅₀value, if the OD of product to be detected and negative control ₄₅₀ratio is greater than 2.1, then kind to be detected contains BtCry1 toxoid.